Understanding the mechanism of Piwi-induced transcriptional silencing Project summary Maintaining genomic integrity is crucial for the survival of all species. Piwi proteins and their small RNA partners, the piwi-interacting RNAs (piRNAs), repress transposon activity in the germline to ensure genomic integrity. In the cytoplasm of germline cells, piwi proteins - guided by piRNAs - recognize and cleave transposon mRNAs. We have recently shown that the Piwi protein is active in the nuclei of Drosophila cells: Piwi silences transposons by inducing transcriptional repression and establishing a repressive chromatin state at its genomic targets. Neither the mechanism of Piwi-induced transcriptional silencing nor the components of the molecular machinery involved in this process downstream of Piwi is known. In this proposal we will comprehensively investigate the mechanism of Piwi-induced transcriptional repression. We will comprehensively characterize changes in chromatin marks and chromatin factors. These experiments will direct further analyses to identify factors responsible for Piwi-mediated transcriptional repression. We will also establish a reporter system for piRNA-independent recruitment of Piwi to specific sites on the genome. We will use this reporter system to identify factors that are required for Piwi-mediated transcriptional silencing. We will also evaluate the role of the N-terminal region of Piwi in transcriptional silencing. Piwi proteins and the associate piRNAs are maternally deposited into the embryo and accumulate in the pole plasm that later gives rise to the developing gonads. The functional relevance of the deposited piRNAs is not known. We will test the role of the piRNA pathway in transgenerational epigenetic silencing in the somatic and germline tissues of the offspring. The proposed studies will significantly advance our knowledge of RNA-mediated regulation of chromatin structure and transcriptional activity in animals. Mechanistic insights into Piwi-mediated transcriptional repression and an understanding of how these processes are involved in transmitting epigenetic information to the progeny will aid in the design of targeted treatment of human sterility caused by deregulation of the Piwi- piRNA pathway.